p53 inactivation and cancer. The tumor suppressor gene p53 is of central importance for the genetic stability of human cells (Donehower and Bradley, 1993; Haffner and Oren, 1995; Gottlieb and Oren, 1996; Ko and Prives, 1996; Hansen and Oren, 1997; Levine, 1997). The p53 protein is active as a homo-tetramer and exerts its tumor suppressor function mainly as a transcription factor that induces G1 and G2 cell cycle arrest and/or apoptosis (Donehower and Bradley, 1993; Haffner and Oren, 1995; Gottlieb and Oren, 1996; Ko and Prives, 1996; Hansen and Oren, 1997; Levine, 1997; Hermeking et al., 1998). The p53-mediated G1 arrest is its best characterized activity and involves transcriptional activation of the downstream gene p21WAF1/CIP1/SDI1 (Haffner and Oren, 1995; Gottlieb and Oren, 1996; Ko and Prives, 1996; Hansen and Oren, 1997; Levine, 1997). Other downstream effector genes for p53-mediated G1 arrest may exist, since p21−/− mouse embryonic fibroblasts do not show complete abrogation of G1 arrest after DNA damage (Brugarolas et al., 1995; Deng et al., 1995). The G2/M block mediated by p53 involves, at least in part, induction of 14-3-3σ (Hermeking et al., 1998).
The mechanisms for apoptosis induction and their relative importance remain less clear at present. In certain settings p53 clearly induces pro-apoptotic genes. These include BAX and Fas/APO1 (Miyashita and Reed, 1995; Owen-Schaub et al., 1995) neither of which, however, is an absolute requirement for p53-induced apoptosis (Knudson et al., 1995; Fuchs et al., 1997; Yin et al., 1997). Recently, many more genes have been identified that are induced directly or indirectly during p53-mediated apoptosis (Polyak et al., 1997; Wu et al., 1997; Yin et al., 1998), but the essential genes for p53-induced apoptosis still have to be determined. Transcriptional repression of anti-apoptotic genes, such as bcl-2, may play a role (Haldar et al., 1994; Miyashita et al., 1994) and other non-transcriptional mechanisms may be important as well (Caelles et al., 1994; Wagner et al., 1994; Haupt et al., 1995; Wang et al., 1996; White, 1996).
Several upstream signals activate p53. These include DNA damage, hypoxia and critically low ribonucleoside triphosphate pools (Kastan et al., 1991; Graeber et al., 1996; Linke et al., 1996). Once activated, p53 induces either cell cycle arrest or apoptosis, depending on several factors such as the amount of DNA damage, cell type and cellular milieu, e.g., presence or absence of growth factors (Donehower and Bradley, 1993; Haffner and Oren, 1995; Gottlieb and Oren, 1996; Ko and Prives, 1996; Hansen and Oren, 1997; Levine, 1997).
Cancer cells show decreased fidelity in replicating their DNA, often resulting in DNA damage, and tumor masses have inadequate neovascularization leading to ribonucleoside triphosphate or oxygen deprivation, all upstream signals that activate p53. In view of p53's capability to induce cell cycle arrest or apoptosis under these conditions it is not surprising that absent or significantly reduced activity of the tumor suppressor protein p53 is a characteristic of more than half of all human cancers (Hollstein et al., 1991; Harris and Hollstein, 1993; Greenblatt et al., 1994). In the majority of cancers, p53 inactivation is caused by missense mutations in one p53 allele, often with concomitant loss-of-heterozygosity (Michalovitz et al., 1991; Vogelstein and Kinzler, 1992; Donehower and Bradley, 1993; Levine, 1997). These mutations affect almost exclusively the core DNA-binding domain of p53 that is responsible for making contacts with p53 DNA-binding sites (Cho et al., 1994), while mutations in the N-terminal transactivation domain or the C-terminal tetramerization domain are extremely rare (FIG. 1) (Beroud and Soussi, 1998; Cariello et al., 1998; Hainaut et al., 1998). Contrary to wild-type p53, p53 cancer mutants have a long half-life and accumulate to high levels in cancer cells (Donehower and Bradley, 1993; Lowe, 1995). This may be explained by their inability to activate the MDM-2 gene (Lane and Hall, 1997), since mdm-2 induces degradation of p53 via the ubiquitin pathway as part of a negative feedback loop (Haupt et al., 1997; Kubbutat et al., 1997). The unusually high frequency of p53 missense mutations in human cancers (as opposed to mutations resulting in truncated proteins) is explained by their dominant-negative effect that depends on the intact C-terminal tetramerization domain. The C-terminus allows p53 cancer mutants to form hetero-tetramers with wild-type p53 (Milner and Medcalf, 1991), thus reducing, or even abrogating, the activity of wild-type p53 protein (Michalovitz et al., 1991; Vogelstein and Kinzler, 1992; Hann, 1995; Brachmann et al., 1996; Ko and Prives, 1996). In addition, there is evidence that at least some of the same missense mutations may confer a gain-of-function (Gottlieb and Oren, 1996; Ko and Prives, 1996; Levine, 1997).
p53 abnormalities and cancer therapy. Considering the activities of the p53 tumor suppressor protein, reconstitution of wild-type p53 activity to cancers would be of large therapeutic benefit, an idea that is supported by several lines of evidence from epidemiological, clinical and basic cancer research (Fisher, 1994; Lowe, 1995; Harris, 1996a).
Several human malignancies that are usually diagnosed at a young age, such as testis cancer, pediatric acute lymphoblastic leukemia and Wilms tumor, can be successfully eradicated even at advanced stages. They all have in common that they carry wild-type p53 (Heimdal et al., 1993; Wada et al., 1993; Malkin et al., 1994). At the same time, subgroups of these malignancies with a poor prognosis, for example the anaplastic variant of Wilms tumor, commonly do carry p53 mutations (Bardeesy et al., 1995). Similarly, tumor types that are often resistant to conventional therapies and difficult to treat at advanced stages, such as lung, prostate, colorectal, breast, head and neck, pancreatic and gastric cancers, show a high frequency of p53 mutations (Hollstein et al., 1991; Fisher, 1994; Lowe, 1995; Harris, 1996a; Beroud and Soussi, 1998; Cariello et al., 1998; Hainaut et al., 1998).
These findings have spurred great interest in exploring p53 as a predictive marker for response to therapy and for overall prognosis. The majority of cancer types have been evaluated to some extent, and the publications are too numerous to be summarized here. As an example, studies in breast, head and neck, lung and ovarian cancers have found a good correlation between p53 abnormalities and poor survival and poor response to therapy (Thor et al., 1992; Allred et al., 1993; Bergh et al., 1995; Rusch et al., 1995; Sauter et al., 1995; Righetti et al., 1996; Bems et al., 1998; Huang et al., 1998). The results are not always unequivocal, as some studies were unable to detect a statistically significant difference between cancers with and without functional p53 (Isola et al., 1992; Elledge et al., 1995). These discrepancies may be due to confounding factors. For example, a cancer with a poor prognosis because of degradation of p53 by overexpressed mdm-2 may be incorrectly scored as a cancer with functional p53 if the mdm-2 status of the cancer is not evaluated. In addition, the sample size of many studies was not large enough to make firm conclusions.
Strong evidence for a central role of p53-mediated apoptosis in cancer therapy is provided by experiments in cell lines with and without functional p53. Comparison of wild-type and p53-deficient thymocytes established that p53 is required for radiation- and etoposide-induced apoptosis (Clarke et al., 1993; Lowe et al., 1993a). Similar experiments in adenovirus E1A transformed mouse embryo fibroblasts showed that apoptosis induced by radiation, 5-fluorouracil, etoposide and adriamycin also depends on functional p53 in these cells (Lowe et al., 1993b). These studies were extended into a mouse model where again only tumors with functional p53 showed good treatment responses to radiation and adriamycin, while p53-negative tumors were highly resistant to therapy and showed little evidence of apoptosis (Lowe et al., 1994). Results of the Developmental Therapeutics Program of the NCI impressively and independently confirmed these findings. An analysis of the cytostatic and cytotoxic effects of 123 compounds on 60 different human cancer cell lines showed a very good correlation between p53 mutations and resistance to many commonly used chemotherapeutic agents (O'Connor et al., 1997; Weinstein et al., 1997). All these data do not necessarily indicate that functional p53 is absolutely essential for chemotherapy-induced apoptosis. In fact, chemotherapy drugs can kill cancer cells through p53-independent mechanisms (Kaufmann, 1989; Strasser et al., 1994; Bracey et al., 1995). The sum of the evidence, however, suggests that cancer agents are significantly more effective in the presence of p53 (Fisher, 1994; Lowe, 1995; Harris, 1996a).
Based on the discussed studies and the general knowledge about p53, p53 and its pathways have been recognized as a prime target for developing new cancer therapies (Fisher, 1994; Gibbs and Oliff, 1994; Kinzler and Vogelstein, 1994; Lowe, 1995; Milner, 1995; Harris, 1996a). In particular, the high frequency of p53 mutations in cancers makes therapeutic strategies for restoring this tumor suppressor pathway highly desirable since a large number of patients could potentially benefit. It has been estimated that every year approximately 330,000 patients in the United States and 2.4 million patients worldwide are diagnosed with cancers that contain p53 mutations (Harris, 1996a, 1996b).
Strategies to partially or completely restore wild-type p53 function to cancer cells. Restoration of wild-type p53 activity to cancer cells is the most direct way of making cancer cells more susceptible to apoptosis and can be pursued in two ways. The first strategy is to reintroduce wild-type p53, perhaps by gene therapy (Roth et al., 1996; Barinaga, 1997; Nielsen and Maneval, 1998), and does not rely on the p53 status of a given cancer. The current major challenge is efficient and selective targeting of wild-type p53 expression constructs to the cancerous cells (Nielsen and Maneval, 1998). A major drawback of this approach is that it may be less effective for cancers with vast amounts of a dominant-negative p53 cancer mutant. This strategy would be greatly aided by the availability of p53 proteins that are resistant to the dominant-negative effects of p53 cancer mutants and that are superior to wild-type p53 in inducing apoptosis, classes of p53 proteins that to date have not been described.
The second strategy is only possible because of the unique pattern of p53 missense mutations in human cancers and aims at therapeutically exploiting the abundant p53 mutant protein found in many cancers. Since the resulting p53 cancer mutants are full-length proteins each with a structurally altered core domain, but an intact transactivation domain and an intact C-terminal tetramerization domain, one could restore wild-type activity to the p53 cancer mutants in these tumors (Gibbs and Oliff, 1994; Lowe, 1995; Milner, 1995; Harris, 1996a). This can be achieved in at least two ways. One attempt has been to interfere with the extreme C-terminal autoregulatory domain of p53 by using antibodies (Halazonetis and Kandil, 1993; Hupp et al., 1993; Abarzua et al., 1995; Niewolik et al., 1995) or peptides spanning part of this region (Hupp et al., 1995; Abarzua et al., 1996; Selivanova et al., 1997). This strategy presumably activates p53 cancer mutants by blocking the ability of the very C-terminus to fold back onto and inhibit the p53 core domain. It could succeed with p53 cancer mutants that retain residual activity and which only require additional activation to exceed the threshold required for biological effects. However, antibodies and peptides clearly cannot be delivered efficiently to cancer cells in patients (Selivanova et al., 1997). Small molecule compounds with similar effects could overcome this problem, but their design is currently not feasible since the exact structural basis of this negative autoregulation and of its neutralization by antibodies or peptides is not known due to lack of a crystal structure for the full-length p53 protein (Ko and Prives, 1996; Selivanova et al., 1997). In addition, this approach may activate mutant and wild-type p53 proteins indiscriminately, thus possibly causing significant side effects due to inappropriate wild-type p53-induced apoptosis in normal tissues.
A more direct approach is to revert the effects of tumorigenic mutations on the structure and function of the p53 core domain itself by means of small molecules. This strategy is preferable since it is predicted to selectively stabilize p53 cancer mutants. It also holds the promise of restoring function to completely inactive p53 cancer mutants. Restoring the normal configuration to a p53 cancer mutant is considered more challenging than inhibiting the function of a protein by small molecules (Gibbs and Oliff, 1994). However, there are examples: small molecule compounds that bind the central cavity of the hemoglobin tetramer can act as allosteric effectors and stabilize the T state of hemoglobin over the R state (Abraham et al., 1992); and small molecule compounds that stabilize the transthyretin tetramer against dissociation can prevent amyloid fibril formation in vitro (Miroy et al., 1996). Furthermore, the technology of structure-based drug design is steadily advancing so that this challenge may be met (Bohacek et al., 1996; Marrone et al., 1997).
p53 mutations and the p53 core DNA-binding domain. These considerations make it clear that a detailed understanding of the structural consequences of p53 cancer mutations on the p53 core domain is needed. More significantly, stabilizing mechanisms must be identified that can override the deleterious structural effects of p53 cancer mutations.
The crystal structure of the wild-type p53 core domain has given enormous insight into how p53 interacts with its DNA-binding sites (Cho et al., 1994). The structures of the C-terminal tetramerization domain and of the N-terminal transactivation domain (complexed to mdm-2) have been determined as well (Clore et al., 1994; Jeffrey et al., 1995; Kussie et al., 1996). The structure of the full-length protein as a homo-tetramer, however, is solely based on computer modeling (Jeffrey et al., 1995) and suggests that the core domain functions as a separate entity that is connected to the other domains through flexible linkers. The core domain spans 191 amino acids and consists of a β sandwich that serves as the scaffold for two large loops (termed L2 and L3) and a loop-sheet-helix motif. The loops and the loop-sheet-helix motif form the DNA-binding surface of p53 and provide contacts to the DNA backbone and the edges of the bases (FIG. 1A). This structural organization was considered unique until the recent discovery of p73 made it clear that p53 is actually part of a family of transcription factors (Jost et al., 1997; Kaghad et al., 1997). The vast majority of tumor-derived p53 missense mutations map to this core domain (FIG. 1B) and invariably result in the reduction or loss of DNA-binding. These cancer mutations are predicted to fall into two classes; one class of mutations maps to DNA-contacting residues and eliminates p53-DNA contacts (functional mutations); the other, larger class of mutations probably affects the structural integrity of the DNA-binding domain (structural mutations). These structural defects may range from small structural shifts to the global destabilization and unfolding of the p53 core domain. The most frequent p53 cancer mutations affect amino acids that are part of important structures of the p53 core domain, such as the L3 loop and the loop-sheet-helix motif that provide DNA contacts. However, the high frequency of a mutation does not predict how deleterious its effects on the structural integrity of the core domain are, since the frequency of these mutations is also determined by exogenous carcinogens and endogenous biological processes (Donehower and Bradley, 1993; Greenblatt et al., 1994).
To date, our understanding of the structural consequences of p53 cancer mutations is limited to predictions using the structure of the wild-type p53 core domain, biochemical data (Cho et al., 1994) and experiments with monoclonal antibodies that recognize areas of the p53 core domain that are not accessible in the correctly folded state (Donehower and Bradley, 1993; Gottlieb and Oren, 1996; Levine, 1997). Similarly, very little is known about how the effects of cancer mutations can be overcome.
There is a need in the art for the identification of small molecules and proteins that will restore function to mutant p53 proteins. Such small molecules and proteins will increase the ability of mutant p53 to induce cell cycle arrest and/or apoptosis. There is also a need in the art for reagents to aid in the development and identification of such p53 suppressors.